Phosphatidyl oligoglycerols

ABSTRACT

In order to form liposomes with a longer half-life in blood, use is made of defined compounds with the general formula (A)

The invention relates to phosphatidyl compounds which contain a definedhydrophilic residue, and to long-circulating liposomes.

Conventional liposomes circulate in the serum for up to 5 hours.However, especially when liposomes are used as a means of drug delivery,it is desirable that they circulate in the bloodstream for as long aspossible.

To this end, the so-called “stealth liposomes” were developed, which arenot destroyed in the bloodstream so quickly. These “stealth liposomes”are built up on the basis of phosphatidyl compounds which have anextended polyethylene glycol residue. The polyethylene glycol residueproved to be most effective in producing the desired increase inliposome survival duration when the molecular weight was between 2000and 3000. A serious disadvantage, however, of these “stealth-liposomes”,ie, of these phosphatidyl compounds with a polyethylene glycol residue,is that the compounds are not exactly defined, since the polyethyleneglycol residues display different chain lengths.

Maruyama et al. (Int. J. Pharmac. 111 (1994), 103-107) suggested the useof dipalmitoyl phosphatidyl polyglycerols to lengthen the duration ofliposome circulation. However, since technical-grade polyglycerols wereused as starting material, no uniform products were obtained hereeither. Technical polyglycerols, which consist of a mixture ofpolyglycerols with different chain lengths and monoglycerol, and whichare characterized by their average molecular weight, werephosphatidylated by means of phospholipase D. The resulting productsonly led to a small increase in the survival duration of liposomes inthe blood.

The object of this invention was thus to provide compounds whichincrease the survival duration of liposomes and which are of exactlydefinable composition.

This objective is established according to the invention by means of acompound with the general formula (A)

-   where R¹ and R², independent of each other, stand for hydrogen or a    saturated or unsaturated alkyl or acyl residue, which may be    branched and/or substituted, R³ stands for hydrogen or an alkyl    residue,-   n=0 or 1,-   x is a whole number from 1 to 4 and-   m is a whole number from 2 to 10 if n=0, or a whole number from 1 to    10 if n=1, or 1 if x is greater than 1,    and where, in the case that n=0, the compound is more than 90%    uniform with respect to the value of m.

The stepwise synthesis—upon which this invention is based—of thehydrophilic residues of the phosphatidyl compounds of formula (A) makesit possible to obtain compounds of an exactly defined composition.

The compound of the invention, with the formula (A), is thus not amixture of various molecules of indefinite composition and chain length,but can be synthesized to have precisely the desired structure. If, forexample, the desired product is a triglycerol derivative, ie, x=1 andm=3 in formula (A), the content of monoglycerol, diglycerol,tetraglycerol and higher oligoglycerol derivatives will be low. It ispreferable if a glycerol derivative of a certain chain length isobtained that is largely free of glycerol derivatives of other chainlengths. The content of monoglycerol derivatives, in particular, is low,amounting to less than 5%, preferably less than 1% or, even morepreferably, less than 0.1% relative to the desired oligoglycerolderivative.

According to the invention, the compound of formula (A) is a uniformcompound of defined structure. It is of advantage if, with respect tothe value of m, the compound is greater than 95% uniform. It is togreater advantage if it is more than 99% uniform. It is even possible toprovide a compound which is more than 99.9% uniform with respect to thevalue of m.

The compound is preferably an oligoglycerol derivative with 2 to 5glycerol units, more preferably with 2 to 4 glycerol units. It is toadvantage if these are 1,3-linked linear oligoglycerol residues.

According to the invention, the residues R¹ and R², independent of eachother, stand for hydrogen, a saturated or unsaturated C₁-C₂₄ alkyl orC₁-C₂₄ acyl residue, preferably hydrogen or a saturated or unsaturatedC₈-C₂₄ alkyl or C₈-C₂₄ acyl residue, it being beneficial if it at leastone of the residues R¹ and R² is an acyl residue.

The residue R³ is preferably hydrogen or an alkyl residue with 1 to 4carbon atoms.

The compound of formula (A) can be a racemic compound which contains aphospho-rac-(1 or 3)-oligoglycerol linkage, or it can be in the form ofa stereospecific isomer. The stereoisomers can have aphospho-sn-1-oligoglycerol linkage or a phospho-sn-3-oligoglycerollinkage. The formation of the stereospecific linkage can be carried outin a manner analogous to those described in the literature (DE 31 30 867A1; H. Eibl et al., Chem. Phys. Lipids, 28 (1981), 1-5, 41 (1986), 53-63and 47 (1988, 47-53).

The subject matter of the invention also includes liposomes whichcontain phospholipids and/or alkyl phospholipids, maybe cholesterol, and1 to 50 mol % of a compound with the general formula (A),

or salts thereof, where the cholesterol, the phospholipids, the alkylphospholipids and the compound of formula (A) together make up 100 mol%, and R¹ and R², independent of each other, stand for hydrogen, asaturated or unsaturated alkyl or acyl residue which may be branchedand/or substituted,

-   R³ stands for hydrogen or an alkyl residue,-   n=0 or 1,-   x is a whole number from 1 to 4 and-   m is a whole number from 2 to 10 if n=0, or a whole number from 1 to    10 if n=1, or 1 if x is greater than 1, and where, in the case that    n=0, the compound (A) is more than 90% uniform in respect of the    value of m.

The liposomes of the invention have a half-life in serum of up to 18 to20 hours. Surprisingly, the liposome concentration in blood was found todecrease linearly.

It is beneficial according to the invention if compound (A) displays auniformity of more than 95% or, even better, more than 99% with respectto the value of m. It is also possible, however, to use compound (A) inpractically pure form, ie, more than 99.9% uniform with respect to thevalue of m.

The liposomes preferably contain a compound of formula (A), in which x=1and m is a whole number from 2 to 5; it is even more preferable if m isa whole number from 2 to 4.

The residues R¹ and R² of the compound of formula (A) contained in theliposomes can stand, independent of each other, for hydrogen or asaturated or unsaturated C₁-C₂₄ alkyl or C₁-C₂₄ acyl residue, preferablyhydrogen or a saturated or unsaturated C₅-C₂₄ alkyl or C₈-C₂₄ acylresidue. The substituent is a residue which does not interfere duringthe preparation. R³ is preferably hydrogen or a C₁-C₄ alkyl residue.

The compound of formula (A) can be present in the liposomes as a racemicmixture, ie, with a phospho-rac-(1 or 3)-oligoglycerol linkage. It ispreferable if it is present in stereospecific form with aphospho-sn-1-oligoglycerol linkage or a phospho-sn-3-oligoglycerollinkage.

It is to advantage if at least one of the residues R¹ and R² of formula(A) is an acyl group.

It is beneficial if liposomes containing the compound of formula (A)with n=0 exhibit an excess negative charge. However, liposomes can alsobe prepared from compounds of formula (A) in which n=1. In this case, itis better if the liposomes exhibit no excess charge or a positive one.

Besides a compound of formula (A), the liposomes contain phospholipidsand/or alkyl phospholipids and maybe cholesterol. It is preferable touse the compound of formula (A) in an amount of 5 to 15 mol %. If theliposomes do not display an excess charge, a composition of 0 to 70 mol% cholesterol, 1 to 50 mol % of a compound of formula (A), andphospholipids and/or alkyl phospholipids is preferred. If there is anegative excess charge, a preferred liposome composition consists of 0to 70 mol % cholesterol, 1 to 15 mol % of a compound of formula (A), andphospholipids and/or alkyl phospholipids. A higher proportion ofcompounds of formula (A) with a negative excess charge would lead toinstability of the liposomes in the blood circulation. It is toadvantage if the liposomes comprise 35 to 43 mol %, in particular 38 to42 mol % cholesterol, 5 to 15 mol % of a compound of formula (A), andphospholipids and/or alkyl phospholipids.

The phospholipids and/or alkyl phospholipids can, for example, be diacylphosphoglycerols of defined structure. Generally speaking, these lipidcomponents can be used as compounds of defined structure.

In the case that x>1, it is preferable if the residue—CH₂(—CHOH)_(x)—CH₂—OH derives from sugar alcohols which have fourhydroxyl groups for x=2, five hydroxyl groups for x=3, and 6 hydroxylgroups for x=4. Examples of such residues are mannitol derivatives forx=4, lyxitol derivatives for x=3 and threitol derivatives for x=2.

The liposomes of the invention have a markedly longer half-life in theblood stream. Their half-life is preferably at least 10 hours, betterstill, more than 12 hours. Half-lives of 18 to 20 hours have beenmeasured for the liposomes of the invention. Surprisingly, the decreasein blood lipid concentration with time was found to be absolutelylinear. It is preferable according to the invention if, after 6 hours,more than 50% of the liposomes added are still present in the blood; itis even more preferable if more than 60% are still present.

A particularly surprising property of the liposomes of the invention istheir preferred tendency to accumulate in the spleen. Depending on thecomposition and size of the liposomes, enrichment thereof in the spleenhas been found which exceeds enrichment in the liver by a factor of 25.Enrichment in the spleen compared with that in the liver increases withincreasing value of m in formula A and with increasing size of theliposomes. With the transition from SUVs (Small Unilamellar Liposomes;diameter about 60 nm) to LUVs (Large Unilamellar Liposomes; diameterabout 190 nm), the degree of enrichment in the spleen increases manytimes over. The preferential accumulation in the spleen also increasesas the number of carbon atoms in R¹ and R² increases.

It was found, in addition, that the liposomes of the invention alsoaccumulate in certain tumour tissues. This was observed to be the case,for example, with breast carcinomas induced by nitrosomethylurea (MNUcarcinoma).

The liposomes of the invention can also contain one or morepharmaceutical drugs.

Generally speaking, all drugs can be used that can be introduced intothe plasma by means of liposomes. Preferred groups of drugs are, on theone hand, cytostatic agents, especially anthracycline antibiotics suchas doxorubicin, epirubicin and daunomycin, with doxorubicin beingespecially preferred. Other preferred anti-tumour drugs are idarubicin,hexadecylphosphocholine,1-octadecyl-2-methyl-rac-glycero-3-phosphocholine, 5-fluoruracil,cis-platinum complexes such as carboplatin and novantron, andmitomycins.

Other preferred groups of drugs are immunomodulating substances such ascitokines, of which interferon and, in particular, α-interferon aregiven special preference, antimycotic substances (eg, amphotericin B),and drugs to combat protozoan diseases (malaria and trypanosome andleishmania infections). Taxol is another preferred drug.

Yet another group of preferred drugs is the group of lytic drugs, as aredescribed in the DE 41 32 345 A1. The content of this patent applicationis thus included by way of reference. Preferred drugs are miltefosin,edelfosin, ilmofosin and SRI162-834.

The subject matter of the invention thus includes use of the liposomesaccording to the invention for preparing an anti-tumour agent, with thedrug doxorubicin being given special preference.

The subject matter of the invention also includes use of the liposomesaccording to the invention for preparing an agent to influence cellproliferation, with the drug preferably being a cytokine, in particularα-interferon.

The subject matter of the invention includes, in addition, apharmaceutical composition to which contains the liposomes describedabove and, entrapped in the liposomes, one or more pharmaceutical drugs,combined if necessary with standard pharmaceutical diluents, adjuvants,carrier media and fillers.

The liposomes of the invention are prepared using methods which areknown per se and with the usual equipment. Typically, a solutioncontaining the various components of the liposome and 1 to 50 mol % of acompound of formula (A) is converted into a lipid suspension which isthen pressed under high pressure through nozzles or a perforated disk;the size of the liposomes can be regulated by means of the size of theperforations in the disk. Suitable measures for converting a lipidsuspension into liposomes are familiar to persons versed in the art.Preferably, 5 to 15 mol % of a compound of the general formula (A), 35to 43 mol % cholesterol and 42 to 60 mol % phospholipids and/or alkylphospholipids are converted into a lipid suspension, which in turn isconverted into liposomes by means of suitable measures and in a mannerknown per se.

These known methods can also be used to make a pharmaceuticalformulation which contains the liposomes of the invention and one ormore pharmaceutical drugs. To entrap water-insoluble drugs, the drug isdissolved together with the lipid components, while to entrapwater-soluble drugs, an aqueous solution which contains thewater-soluble drug is added to the lipid film.

The compounds of the invention, having the formula (A), can be preparedin cases where n=1 by linking a defined oligoglycerol with aphosphatidyl ethanolamine by way of the amino group. This results inneutral compounds, ie, compounds without an excess charge. The definedoligoglycerols used for linking are compounds with the formula (B).

In cases where n=0, compounds with the general formula (A) are made bylinking a defined oligoglycerol with a phosphatidylglycerol. When n=0,compounds with the general formula (A) can also be made—using aphosphorylation agent—by linking a defined oligogycerol or a C₄-C₆ sugaralcohol with an alcohol of the formula CH₂—OR¹—CHOR²—CHOH. Asphosphorylation agent, use is made preferably of POCl₃.

The preparation of phospholipids from diacyl glycerols is described inthe literature (DE 32 39 817 A1; P. Woolley et al., Chem. Phys. Lipids47 (1988), 55-62; H. Eibl et al., Chem. Phys. Lipids 47 (1988), 63-68),and this method can be applied here.

Using the above-described methods, a racemic compound is formed whichcontains a phospho-rac-(1 or 3)-oligoglycerol linkage. It is toadvantage if stereospecific compounds are formed, which exhibit aphospho-sn-1-oligoglycerol linkage or a phospho-sn-3-oligoglycerollinkage. To make a compound of formula (A), it is preferable to use alinear oligoglycerol of defined chain length.

The subject matter of the invention also includes a protectedoligoglycerol of the formula (B),

where Y is a whole number from 1 to 9 and X is a benzyl, alkyl ortetrahydropropanyl group. It is beneficial if Y is a whole number from 1to 3. It is possible according to the invention to obtain 1,3-linkedoligoglycerols in practically pure form. Oligoglycerols of a predefinedchain length can be prepared which contain hardly any impurities in theform of oligoglycerols with different chain lengths. In addition, theseoligoglyerols of the invention are practically free of monomericglycerol. In other words, uniform compounds are obtained, which have adefined structure.

In the oligoglycerol, X can also stand for a different suitableprotective group. It is also possible to replace the acetone withanother protective group, in particular another ketone.

The invention comprises, in addition, alkyl oligoglycerols of formula(C)

where Y is a whole number from 0 to 8, preferably a whole number from 1to 3, and one of the residues X or Z is a saturated or unsaturated alkylresidue and the other residue is hydrogen. These alkyl oligoglycerolsare also uniform compounds of defined structure.

The production of oligoglycerols, protected oligoglycerols and alkyloligoglycerols is of particular interest, because with the help of thesestarting materials a number of important and novel adjuvants serving assolubilizers and to improve membrane permeation are obtained. Ofparticular interest with respect to increasing the period for which theliposomes survive in the blood stream is the production of phosphatidyloligoglycerol derivatives of formula (A), which carry additionalhydroxyl groups in the polar area.

Due to the preferred enrichment of the liposomes of the invention in thespleen, these liposomes are suitable generally for the selectiveintroduction of substances into the spleen. These substances may bemedicinal products, contrast agents or the like. This is especiallyimportant with regard to improving the quality of vaccines, since thespleen plays a major role in the formation of antibodies for the immunesystem. In the same way, the enrichment of the liposomes according tothe invention such as was observed in tumour tissue is of importancewith regard to delivering drugs, contrast agents and the likespecifically to such tissue.

The following examples, together with the enclosed drawings, explain theinvention in more detail. In the drawings:

FIG. 1 shows the total-organ distribution of liposomes according to theinvention in the spleen and in the liver.

FIG. 2 shows the per-gram distribution of liposomes according to theinvention in the spleen and in the liver.

FIG. 3 shows how the blood levels of different liposomes of theinvention vary as a function of time.

EXAMPLE 1

In an animal experiment, liposomes were used which consisted of 40 mol %cholesterol, 10 mol % phosphatidylglycerol and 50% dipalmitoyl lecithin.The liposomes had a half-life in serum of 4 hours, with a typicalpersistence characteristic, ie, a rapid decrease to start with, followedby a slower decrease.

Liposomes of the same composition were prepared, in which thephosphatidylglycerol was replaced by a phosphatidylglycerol G2 of theinvention. A half-life in serum of 18 to 20 hours was measured, thedecrease with time being absolutely linear. This linear relation wasobserved irrespective of the size of the liposomes. The same linearreduction in serum liposome concentration was found with 50 nm liposomesand with 150 nm liposomes. The linear reduction in blood liposomeconcentration was also observed for different starting concentrations.

EXAMPLE 2 Percentage of Liposomes in the Blood Stream after 6 Hours

Liposomes according to the invention were prepared, consisting ofdipalmitoyl-sn-G-3-PC/cholesterol/dipalmitoyl-sn-G-3-PG_(Y) in a molarratio of 45:45:10. The percentages of liposomes still in the blood after6 hours are listed in Table 1. For comparison, the percentages measuredby Maruyama et al. under the same conditions for the systemdistearyl-sn-G-3-PC/cholesterolidipalmitoyl-sn-G-3-PG_(Yi) 45:45:10 arelisted too. Compared to the prior art, the example of the inventionshows a pronounced increase in the quantity of liposomes found.

TABLE 1 Y Comparative example Y Example of the invention 0 18% 0 21% 219% 2 80% 3 — 3 82% 4 20% 4 56%

EXAMPLE 3

Liposomes consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphoglyceroglycerol (PG_(n)) andcholesterol in a molar ratio of 4:1:5 were doped with tritium-labelledinulin. These liposomes were administered to rats in a dosage of 100μmol lipid per kg rat, and after 72 hours the distribution of theseliposomes in the spleen and the liver was determined by measuring theradioactivity. Liver weights varied between 9 and 10 g, those of thespleen between 0.6 and 0.7 g. FIG. 1 of the enclosed drawings shows thatfor a liver weight which is about 15 times higher than that of thespleen, the distribution of liposomes (SUVs) increases substantially infavour of the spleen as the number of glycerol units increases (x=1; m=1to 4 in formula A).

In FIG. 2, the liposome uptake by the spleen and the liver is depictedas uptake per gram of the organ. For n=4, the spleen is seen to have aliposome concentration which is about 9 times higher than that of theliver; for n=1, the enrichment factor equals 4. In the last column,FIGS. 1 and 2 show the effect which the size of the liposomes has. ForLUVs with a diameter of 190 nm, liposome enrichment is even more infavour of the spleen: even when n only equals 2, the enrichment factorequals 24. In practical terms, this means that it is no longer possibleto target the liver with these liposomes.

EXAMPLE 4 Preparation of Compounds with the Formula (A) EXAMPLE 4a KeyIntermediate with the Formula I

The oligoglycerols diglycerol (G₂), triglycerol (G₃) and tetraglycerol(G₄) can be prepared from an easily obtained key intermediate with theformula I, 1,2-isopropylidene-rac-glycero-3,1-rac-glycero-3-allylether,(see model A).

Model a: Oligoglycerols from Formula I

The intermediate product described by formula I can be obtained in largequantities from commercial allyl glycidyl ether by NaOH-catalyzed ringopening with 1,2-isopropylidene-rac-glycerol, which is likewiseavailable in the chemicals trade:

Epoxide Opening with Alcohols (General Example) Production of the KeyIntermediate with the Formula I:1,2-isopropylidene-rac-G₁-3,1-0,0-3-0-allyl-rac-G₂

A catalytic quantity of NaOH (MW 40.00; 0.6 mol-24 g) is added to1,2-isopropylidene-rac-glycerol (MW 132.16; 16 mol-2115 g), which isrendered a solution by stirring and heating to 80° C. At 80° C., allylglycidyl ether (MW 114.14; 6 mol—685 g) is added dropwise over a periodof 2 hours, and the reaction mixture stirred for another 2 hours at 80°C. By this point in time the epoxide (R_(f) in ether=0.8) has reactedcompletely to form the G₃ constituent (R_(f) in ether=0.6) The excessisopropylidene-rac-glycerol has an R_(f) of 0.65 in ether and is removedfrom the reaction mixture at 75° C./10 mbar. The residue has 1 ldiisopropyl ether added to it and is extracted twice with 1 l NaCl (1%solution in H₂O) in each case. The organic phase is rotated in anevaporator and distilled (Kpi₁₀-1 mbar 125° C.).

The yield of the pure product1,2-isopropylidene-rac-G₁-3,10,0-3-0-allyl-rac-G₂ (MW 246.30) is 1025 g(ca. 70%).

Instead of 1,2-isopropylidene-rac-glycerol, it is possible to reactother primary alcohols and also allyl alcohol and benzyl alcohol underthe given conditions. In the same way, it is also possible to use otherepoxides.

The intermediate product with the formula I can also be obtained from1,2-isopropylidene-rac-glycero-3-glycidyl ether by means ofNaOH-catalyzed ring opening with allyl alcohol. In this case,1,2-isopropylidene-rac-glycero-3-glycidyl ether must first be made fromallyl glycerol.

EXAMPLE 4b Alkylation of Primary or Secondary Hydroxyl Groups (GeneralExample) Preparation of a Key Intermediate:1,2-isopropylidene-rac-G₁-3,1-0,0-2-0-benzyl-3-0-allyl-rac-G₂

The key intermediate, 1,2-isopropylidene-rac-G₁-3,1-rac-G₂-0-allyl ether(MW 246.30; 0.5 mol-123 g) is dissolved in 500 ml tetrahydrofuran, hasbenzyl chloride (0.6 mol-76 g) added to it and is reflux-boiled. K-tert.butylate (0/mol-79 g) dissolved in 500 ml tetrahydrofuran is addeddropwise. The reaction is completed after 30 minutes of reflux-boiling(TLC check—R_(f) in ethyl ether: educt, R_(f)=0.1; product, R_(f)=0.4).The reaction mixture has 1 l diisopropyl ether and 1 l 1% NaCl solutionadded to it, is shaken, and the upper phase rotated in an evaporator.The product can either be used directly, or recovered in pure form inapproximately 90% yield by means of chromatography on silica gel.

Empirical formula C₁₉H₂₈O₅ (MW 336.42)

calculated: C, 67.83; H, 8.39; O, 23.79.

measured: C, 67.78; H, 8.34; O, -.

Instead of benzyl chloride, use can also be made of benzyl bromide,allyl chloride or allyl bromide, or of the halogenides or mesylates ofprimary alcohols. The products of the reaction between primary orsecondary hydroxyl groups and alkyl mesylates, in particular, lead tohigh yields (>90%) of the desired target compounds.

EXAMPLE 4c Synthesis Sequence 0-allyl ether→0-propenyl ether→alcohol(General Example) Preparation of2-0-benzyl-rac-G₁-1,3-0,0-1,2-isopropylidene-rac-G₂

Rearrangement

1,2-isopropylidene-rac-G₁-3,1-0,0-2-0-benzyl-3-0-allyl-rac-G₂ (0.5mol-168 g) is dissolved in 500 ml DMF, to which k-tert. butylate (0.7mol-79 g) is then added. The reaction mixture is heated to 110 to 115°C. with continuous stirring, left for 15 minutes at this temperature andthen cooled to 20° C. Following the addition of 500 ml diisopropyl etherand 500 ml 1% NaCl, the upper, diisopropyl ether phase is removed andthe solvent eliminated under vacuum (TLC check—R_(f) inhexane/diisopropyl ether (1:1): educt, R_(f)=0.2; product, R_(f)=0.4).

Cleavage of the Propenyl Protective Group

The residue from the above reaction, approximately 168 g, is dissolvedin 500 ml methanol and, following addition of 50 ml 1 M HCl, isreflux-boiled. The reaction is complete after 60 minutes (TLC check inhexane/diisopropyl ether (1:1): educt, R_(f)=0.4; product, R_(f)=0). Theyield of rac-G₁-3,1-rac-G₂-2-0-benzyl ether is >90%. Under the acidicconditions prevailing during propenyl cleavage, the isopropylideneprotective group is likewise removed. If necessary, it can bereintroduced in the 1.2 position.

Introduction of the Isopropylidene Protective Group

The residue from the above reaction (approx. 0.5 mol) is dissolved in300 ml THF to which, in succession, 2,2-dimethoxypropane (0.5 mol-52 g)and 0.2 g H₂SO₄ in 10 ml THF are added, and then stirred for 2 hours at25° C. The reaction mixture is neutralized with saturated Na₂CO₃solution, the precipitate removed under suction and the filtrate rotatedwith xylol under vacuum to free it of water. The product is purifiedchromatographically on silica gel 60 (Merck, grain size 0.2-0.5 mm)(R_(f) in diethyl ether: educt, R_(f)=0.0; product, R_(f)=0.4). Oneobtains 121 g of the important intermediate needed for the preparationof phosphatidyl-diglycerols (G₂ parent substance).

G₂ Parent System: 2-0-benzyl-rac-G₁-1,3-0,0-1,2-isopropylidene-rac-G₂(yield 82%).

Empirical formula: C₁₆H₂₄O₅ (MW 296.36)

calculated: C, 64.85; H, 8.16; O, 26.99.

measured: C, 64.82; H, 8.14; O, -.

Intermediates which have higher proportions of oligoglycerol and arelikewise needed for the production of phosphatidyl oligoglycerols can beprepared analogously. Some analytical findings pertaining to keyintermediates are summarized below:

G₃ Parent System:2-0-benzyl-rac-G₁-1,3-0,0-2-0-benzyl-rac-G₂-1,3-0,0-1,2-isopropylidene-rac-G₃

Empirical formula: C₂H₃₆O₇ (MW 460.56)

calculated: C, 67.81; H, 7.88; O, 24.32.

measured: C, 67.75; H, 7.85; O, -.

G₄ Parent System:

-   2-0-benzyl-rac-G₁-[1,3-0,0-2-O-benzyl-G]₂-1,3-0,0-1,2-isopropylidene-rac-G₄

Empirical formula: C₃₆H₄₈O₉ (MW 624.77)

calculated: C, 69.21; H, 7.74; O, 23.05.

measured: C, 69.17; H, 7.69; O, -.

G₆ Parent System:

-   2-0-benzyl-rac-G_(1[)1,3-0,0-2-0-benzyl-rac-G]₄-1,3-0,0-1,2-isopropylidene-rac-G₆

Empirical formula: C₆₆H₇₂O₁₃ (MW 953.172)

calculated: C, 70.57; H, 7.61; O, 21.82.

measured: C, 70.56; H, 7.54; O, -.

G₈ Parent System:

-   2-0-benzyl-rac-G₁-[1,3-0,0-2-0-benzyl-rac-G]₆-1,3-0,0-1,2-isopropylidene-rac-G₈

Empirical formula: C₇₆H₉₆O₁₇ (MW 1281.58)

calculated: C, 71.23; H, 7.55; O, 21.22.

measured: C, 71.15; H, 7.53; O, -.

EXAMPLE 4d Substances which Bear the Tetrahydropyranyl Protective Group(Instead of Benzyl)

(Preparation of Phosphatidyl Oligoglycerols which Contain UnsaturatedFatty Adds)

For this variant, 1,2-isopropylidene-rac-glycero-3-O-allyl ether isprepared and epoxidized as described by H. Eibl and P. Woolley (Chem.Phys. Lipids 41 (1986) 53-63).

Epoxidation (General Example)

1,2-isopropylidene-rac-glycero-3-0-allyl ether (MW 172.22; 1 mol-172 g)is dissolved in 1 l CH₂Cl₂. 3-chloroperoxybenzoic acid (1.1 mol) isadded portion wise and the reaction mixture stirred for 6 hours at25-30° C. The educt (R_(f) 0.5 in diethyl ether/pentane 1:1) is by thentransformed completely into the desired product (R_(f) 0.2 in the abovesystem). After removing the precipitate by suction filtration, 100 gNa₂CO₃ is added to the filtrate and the mixture stirred for another 3hours at 20° C. The precipitate is removed and the solvent eliminatedunder vacuum. The yield of epoxide (MW 188.22) is 170 g (90%). Asdescribed under “epoxide opening with alcohols” (Example 4a), theepoxide is now converted with benzyl alcohol into1-O-benzyl-rac-G₁-3,1-0,0-2,3-isopropylidene-rac-G₂ and the free —OHgroups converted with 3,4-dihydro-2H-pyran into the tetrahydropyranderivative.

Introduction of the Tetrahydropyran Protective Group (General Example)

1-0-benzyl-rac-G₁-3,1-0,0-2,3-isopropylidene-rac-G₂ (MW 296.36; 1mol-296 g) is dissolved in 1 l THF, to which 1.4 mol3,4-dihydro-2H-pyran and 0.1 mol toluene sulfonic acid are added. Thereaction is complete after 1 hour (educt, R_(f) 0.65; product, R_(f)0.90 in diethyl ether). 1 l 0.2 mol Na₂CO₃ solution and 1 l diisopropylether are added, and the mixture shaken thoroughly in a separatingfunnel. The upper phase is rotated in an evaporator and the productconverted by means of hydrogenolysis with H₂ in the presence of a PD/Ccatalyst (5% Pd based on the alcohols) into the G₂ constituent with freehydroxyl group.

G2 Parent System:2-0-tetrahydropyranyl-rac-G₁-1,3-0,0-1,2-isopropylidene-rac-G₂

(Yield 80% expressed in terms of the epoxide)

Empirical formula: C₁₄H₂₇O₆ (MW 291.36)

calculated: C, 57.71; H, 9.34; O, 32.95.

measured: C, 57.59; H, 9.29; O, -.

Compounds with other parent systems can be converted into THP-protectedstructures in the same way. For example, the 3-O-allyl ether of example4a can be converted to an epoxide and opened with allyl alcohol. Again,a 3-O-allyl ether is formed, which is epoxidized and opened with benzylalcohol to form the product below, which, through introduction of 3 THPprotective groups and catalytic hydrogenolysis, can be converted to anintermediate with the G₄ parent system.

G₄ Parent System:

-   2-0-THP-rac-G_(1[)1,3-0,0-2-0-THP-rac-G]₂-1,3-0,0-1,2-isopropylidene-rac-G₄

Empirical formula: C₃₀H₄₅O₂ (MW 607.75)

calculated: C, 59.29; H, 9.12; O, 31.59.

measured: C, 59.24; H, 9.08; O, -.

EXAMPLE 4e Further Processing of the Intermediate with the Formula I G₂Parent System (Racemic)

From formula I, a key intermediate for the preparation of the G₂ parentsystem is obtained (see model B). To this end, the secondary —OHfunction in formula I is alkylated, benzylated, or protected withtetrahydropyran.

Model B: Key Intermediate for Preparing the G₂ Parent System:

X=saturated or unsaturated alkyl, benzyl or THP

Alkyl-G₂Compounds 1) 2-0-alkyl-rac-G₁-1,3-0,0-rac-G₂

The intermediate compound of formula II in which X=alkyl is freed of theprotective groups. The following compounds were isolated:

2-O-ethyl-G₂: C₈H₁₈O₅ (194.23) 2-O-hexyl-G₂: C₁₂H₂₆O₅ (250.33)2-O-undecenyl-G₂: C₁₇H₃₄O₅ (318.45) 2-O-dodecyl-G₂: C₁₈H₃₈O₅ (334.49)2-O-octadecyl-G₂: C₂₄H₅₀O₅ (418.65) 2-O-erucyl-G₂: C₂₈H₅₆O₅ (472.75)

2) 1-O-alkyl-rac-G₁-3,1-0,0-rac-G₂

In the intermediate compound of formula II in which X=benzyl, allyl isremoved from the 1-position and the corresponding alkyl chainincorporated in the 1-position. Following removal of the protectivegroups, the following compounds were obtained:

1-O-methyl-G₂: C₇H₁₆O₅ (180.20) 1-O-propyl-G₂: C₉H₂₀O₅ (208.25)1-O-nonyl-G₂: C₁₅H₃₂O₅ (292.41) 1-O-undecyl-G₂: C₁₇H₃₆O₅ (320.47)1-O-dodecyl-G₂: C₁₈H₃₈O₅ (334.49) 1-O-octadecyl-G₂: C₂₄H₅₀O₅ (418.65)

Unsaturated 1-O-alkyl diglycerols can also be obtained directly by wayof epoxide opening of 1,2-isopropylidene-glycero-glycidyl ether (ModelD, formula IV) with alcohols, eg,

1-O-Undecenyl-G₂: C₁₇H₃₄O₅ (318.45)

However, this path is only suitable for shorter-chain alcohols, sincethe yields for long-chain alcohols such as oleyl alcohol are low. Toprepare 1-oleyl-G₂, therefore, a synthetic pathway via2-O-THP-glycero-1,3-O,O-(1,2-isopropylidene)-glycerol is preferred(model D, formula V)

1-O-Oleyl-G₂: C₂₄H₄₈O₅ (416.64)Intermediates for the Synthesis of Phospholipids which ContainDiglycerols in the Polar Area

Compounds with good protective groups for these syntheses contain a2-O-benzyl ether or a 2-O-tetrahydropyranyl ether group in G₁.

1) 2-O-benyzl-rac-G₁-1,3-0,0-(1,2-isopropylidene)-rac-G₁

C₁₆H₂₄O₅ (296.36)

The compound with the formula III is obtained by alkaline allyl/propylrearrangement, benzylation of the secondary —OH group and subsequentacidic cleavage of the propenyl protective group.

Model C: Starting Product for Phosphatidyl Diglycerols with SaturatedFatty Acid Residues.

2) 2-O-tetrahydropyranyl-rac-G₁-1,3-0,0-(1,2-isopropylidene)-rac-G₂

C₁₄G₂₇O₆ (291.36)

The compound with the formula V is made from allyl glycerol. Theintermediate IV is obtained by way of addition of isopropylidenefollowed by epoxidation. After opening the epoxide with benzyl alcohol,the THP protective group is introduced and the benzyl group removed.

Model D: Starting Product for Phosphatidyl Diglycerols with UnsaturatedFatty Acid Residues G₃ Parent System (Racemic)

From the key intermediate with the formula II it is possible, withinclusion of the allyl group, to develop triglycerols. Followingepoxidation, various intermediates and end products of pharmaceuticalinterest can be made from the epoxide.

Model E: Starting Products for Making G₃ Parent Systems.

Triglycerols can be prepared from the key intermediate with the formulaVI; the intermediate is also used for making G₄ parent systems. Informula VI, X stands for hydrogen, a saturated alkyl, a benzyl or a THPresidue.

Alkyl-G₃ Compounds 1) 1-O-alkyl-rac-G₁-1,3-0,0-rac-G2-1,3-0,0-rac-G₃

The epoxide with the formula VI (X═H) is opened directly with alcoholsand, after the isopropylidene protective group has been split off,results in the following compounds:

1-O-ethyl-G₃: C₁₁H₂₄O₇ (268.30) 1-O-hexyl-G₃: C₁₅H₃₂O₇ (324.41)1-O-nonyl-G₃: C₁₈H₃₈O₇ (366.491) 1-O-undecenyl-G₃: C₂₀H₄₀O₇ (392.53)1-O-dodecyl-G₃: C₂₁H₄₄O₇ (408.57)

For longer-chain alcohols, direct opening results in poor yields. Forthis reason, the oleyl and erucyl compounds of G₃ were prepared byopening of VI (X=THP) with benzyl, THP-protection of the secondaryhydroxyl groups formed, catalytic debenzylation, alkylation in the 1position and removal of the protective groups.

1-O-oleyl-G₃: C₂₇H₅₄O₇ (490.72) 1-O-erucyl-G₃: C₃₁H₆₂O₇ (456.82)

2) 2-O-alkyl-rac-G₁-1,3-0,0-rac-G₇-1,3-0,0-rac-G₃

The epoxide of formula VI (X=benzyl or THP) is opened with allyl alcoholand alkylated in the 2 position. The protective groups are removed inthe usual way. For the preparation of the unsaturated 2-0-alkylcompounds, rearrangement of the allyl protective groups must precedealkylation. In addition, only the THP protective group and not benzylcan be used in G₂ here. The following compounds were prepared:

2-O-methyl-G₃: C₁₀H₂₂O₇ (111.99) 2-O-propyl-G₃: C₁₂H₂₆O₇ (282.33)2-O-nonyl-G₃: C₁₈H₃₈O₇ (366.49) 2-O-undecenyl-G₃: C₂₀H₄₀O₇ (392.53)2-O-dodecyl-G₃: C₂₁H₄₄O₇ (408.57) 2-O-hexadecyl-G₃: C₂₅H₅₂O₇ (464.68)2-O-oleyl-G₃: C₂₇H₅₄O₇ (490.72) 2-O-erucyl-G₃: C₃₁H₆₂O₇ (546.82)Intermediates for the Synthesis of Phospholipids which ContainTriglycerides in the Polar Area

Benzyl and tetrahydropyranyl (THP) residues are convenient protectivegroups for synthesizing phospholipids which have G₃ residues in thepolar area. Benzyl residues are readily removed under mild conditions,provided that only saturated fatty acids are used. THP residues are ofparticular interest because they can be removed in a single steptogether with isopropyl protective groups.

1)2-O-benyzl-rac-G₁-1,3-0,0-(2-O-benzyl)-rac-G₇-1,3-0,0-(1,2-isopropylidene)-rac-G₃

C₂₆H₃₆O₇ (460.56)

This compound is obtained from the key intermediate VI (X=benzyl) byopening with allyl alcohol, benzylation of the 2 position and cleavageof the allyl protective group. In the text, the compound is referred toas formula VII.

2)2-O-THP-rac-G₁-1,3-0,0-(2-0-THP)-rac-G₂-1,3-0,0-(1,2-isopropylidene)-rac-G₃

C₂₂H₄₁O₉ (449.56)

To prepare unsaturated G₃-phospholipids, the residue X=THP is used inVI. The epoxide VI is opened with benzyl alcohol, the secondary hydroxylgroup thus exposed protected with THP, and the benzyl residue removedcatalytically with H₂/Pt. The compound made in this way is referred toin the text as formula VIII.

Additional Remarks

In the description so far we have not made use of the fact that informula VI for X=saturated alkyl, compounds of the following structurecan readily be prepared:1-O-alkyl-rac-G₁-3,1-0,0-(2-0-alkyl)-rac-G₂-3,1-rac-G₃. Therepresentatives of these new structures were made by opening the epoxideVI (X=hexadecyl) with CH₃OH or undecenyl alcohol and splitting off theisopropylidene protective group.

1-O-methyl-rac-G₁-3,1-0,0-(2,0-hexadecyl)-rac-G₂-3,1-rac-G₃

C₂₆H₅₄O₇ (478.71)

1-O-Undecenyl-rac-G₁-3,1-0,0-(2-O-hexadecyl)-rac-G₂-3,1-O,O-rac-G₃

C₃₆H₇₂O₇ (616.958)

G₄ Parent System (Racemic)

G₄ parent systems can be prepared from the key intermediate with theformula IX.

Model F: Starting Products for the Preparation of G₄ Parent Systems.

Tetraglycerols can be made from the key intermediate with the formulaIX. They can also be used to prepare pentaglycerols.

Oligoglycerols with two or more alkyl residues can be made from theintermediates, too. Suitable starting compounds here are molecules withthe formula IX, in which X is a saturated alkyl residue.

Alkyl-G₄ Compounds 1)1-O-alkyl-rac-G₁-1,3-0,0-rac-G₇-1,3-0,0-rac-G₃-1,3-0,0-rac-G₄

The epoxide of formula IX, X═H, is opened directly with alcohols. Afterthe isopropylidene protective group has been split off, the followingsubstances are obtained:

1-0-ethyl-G₄: C₁₄H₃₀O₉ (342.38) 1-0-hexyl-G₄: C₁₈H₃₈O₉ (398.49)1-0-undecyl-G₄: C₁₉H₄₀O₉ (412.52) 1-0-undecenyl-G₄: C₁₉H₃₈O₉ (410.50)1-0-dodecyl-G₄: C₂₀H₄₂O₉ (426.54)

This path is only suitable for shorter-chain alcohols, since the yieldsare much lower with long-chain alcohols.

For long-chain, saturated alcohols it is therefore necessary, as with G₂and G₃, to select a synthetic pathway via the key intermediate withX=benzyl. One opens with allyl alcohol, benzylates the thus exposed 2-OHgroup, removes the allyl group in the 1 position and alkylates the 1position. After removing the protective groups one obtains:

1-0-hexadecyl-G₄: C₂₄H₅₀O₉ (482.99) 1-0-octadecyl-G₄: C₂₆H₅₄O₉ (510.70)1-0-behenyl-G₄: C₃₀H₆₂O₉ (566.81)

2. 2-O-alkyl-rac-G₁-1,3.-0,0-rac-G₉-1,3-0,0-rac-G₃-1,2-0,0-rac-G₄

The key intermediate with the formula IX is opened with allyl alcohol,and the thus-exposed 2 position alkylated. After removal of theprotective groups one obtains:

2-0-propyl-G₄: C₅H₃₂O₉ (356.41) 2-0-hexyl-G₄: C₁₈H₃₈O₉ (398.49)2-0-nonyl-G₄: C₂₁H₄₄O₉ (440.57) 2-0-undenyl-G₄: C₁₉H₃₈O₉ (410.50)2-0-dodecyl-G₄: C₂₀H₄₂O₉ (426.54) 2-0-hexadecyl-G₄: C₂₄H₄₀O₉ (483.99)2-0-octadecyl-G₄: C₂₆H₅₄O₉ (510.70) 2-0-oleyl-G₄: C₂₆H₅₂O₉ (508.69)2-0-erucyl-G₄: C₃₀H₆₀O₉ (564.80)Intermediates for the Synthesis of Phospholipids which haveTetraglycerols in the Polar Area.

As with the synthesis of G₂ and G₃ compounds, benzyl and tetrahydropyranether are suitable protective groups for these syntheses.

1)2-O-benzyl-rac-G₁-1,3-0,0-(2-0-benzyl)-rac-G₂-1,3-0,0-(2-0-benzyl)-rac-G₃-1,3-0,0-(1,2-isopropylidene)-rac-G₄

C₃₆H₄₈O₉ (624.77)

The important intermediate for the synthesis of phospholipids with G₄residues in the polar area is made from formula IX, X=benzyl by openingthe epoxide with allyl alcohol, benzylating the thus exposed 2-OH groupand removing allyl. The compound is referred to as formula X in thetext.

2.2-O-THP-rac-G₁-1,3-0,0-(2-0-THP)-rac-G₂-1,3-0,0-(2-0-THP)-rac-G₃-1,3-0,0-(1,2-isopropylidene)-rac-G₄

C₃₀H₄₅O (607.75)

To make this compound, which is suitable for obtaining unsaturatedphospholipids with G₄ parent systems in the polar area, one proceedsanalogously as for the preparation of the G₃ compound. One opens theepoxide VII, X=THP with benzyl alcohol, protects the thus exposed 2-OHgroup with THP, and removes benzyl with H₂ (Pd/C catalysis.). Thecompound is referred to in the text as formula XI.

Intermediates for the Synthesis of Phospholipids which ContainOligoglycerols in the Polar Area and Permit an SN-1 Linkage to thePhosphate (Natural Configuration)

In the preparation of compounds suitable for incorporation in the polararea of phospholipids (formula III and V for G₂, formula VII and VIIIfor G₃, formula X and XI for G₄), no attention was paid so far to thefact that in natural phosphatidylglycerol, ie, in1,2-diacyl-sn-glycero-3-phospho-sn-1-glycerol, the link betweenphosphate and the non-acylated glycerol is an sn-1 linkage. Since theliposome components, as carriers of medicinal products, should be usedin the most natural configuration possible, synthetic pathways weredeveloped which also permit an sn-1 configuration of the polaroligoglycerol (model G).

sn-1-G₁-G₂ Linkage

The stereospecific linkage can be obtained using methods analogous tothose described in the literature (DE 31 30 867 A1; H. Eibl, Chem. Phys.Lipids 28 (1981) 1-5; H. Eibl et al., Chem. Phys. Lipids 41 (1986)53-63; H. Eibl et al., Chem. Phys. Lipids 47 (1988) 47-53).

The starting product for this linkage is2-0-benzyl-3-O-allyl-sn-glycerol, which, following epoxidation, ishydrolysed to the diol. Following reaction with H⁺/2,2-dimethoxypropane,2-0-Be-sn-G₁-3,1-0,0-(1,2-isopropylidene)-rac-G₂ is obtained, a moleculewith the formula XII, which permits an sn-1 linkage to the phosphategroup in phospholipids and corresponds to the racemate of formula III.

sn-1-G₁-G₂-G₃ Linkage

The starting product for this linkage is again2-0-benzyl-3-O-allyl-sn-glycerol. Protection of the sn-1 position withTHP is followed by epoxidation, and then the epoxide ring opened with1,2-isopropylidene glycerol. The thus-exposed —OH function in G₂ isbenzylated, and the THP protective group removed. One obtains a moleculewith the formula XIII, which permits an sn-1 linkage to the phosphategroup in phospholipids. The molecule XIII corresponds to the racemate offormula VII.

sn-1-G₁-G2-G₃-G₄ Linkage

The starting product is again 2-0-benzyl-3-O-allyl-sn-glycerol, in orderto ensure the sn-1 linkage. Incorporation of the THP protective group isfollowed by epoxidation, and the epoxide then opened with allyl alcohol.Following epoxidation of the intermediate, the epoxide is opened withisopropylidene glycerol, the two exposed —OH groups benzylated, and THPremoved. One obtains XIV, which permits an sn-1 linkage to the phosphateand corresponds to the racemate of formula X.

If desired, compounds with an sn-3-G₁-G₂, sn-3-G₁-G₂-G₃ orsn-3-G₁-G₂-G₃-G₄ linkage with the phosphate can be made analogously. Inthis case, the same sequence of reactions is required, but instead of2-0-benzyl-2-0-allyl-sn-glycerol, use is made of the enantiomeric2-0-benzyl-1-0-allyl-sn-glycerol.

sn-1-G₁-G₂ Linkage

sn-1-G₁-G₂-G₃ Linkage

sn-1-G₁-G₂-G₃-G₄ Linkage

Model G: Phospholipid Constituents which Permit an sn-1-G_(x) Linkage

-   -   (x=2-4). Starting product is 2-O-benzyl-3-O-allyl-sn-glycerol.

EXAMPLE 4f Intermediates which Contain Sugar Alcohols (General Examples)

Important intermediates here are, in particular, such sugar alcohols asare obtainable at a reasonable price or can be made from these by meansof simple reactions (see enclosed table). Of special interest areD-mannitol as open form of inositol, xylitol, which, when the middlecarbon atom is phosphorylated, shows no optical activity and which isreadily obtained as 1,2; 4,5-diisopropylidene xylitol, andmeso-erythritol. The protective groups chiefly employed here areisopropylidene, trityl in combination with benzyl, or allyl.Tetrahydropyranyl is also of some importance as protective group. Somealternatives will now be described which serve as examples.

1.2; 4,5-diisopropylidene-xylitol (General Example for Introduction ofthe Isopropylidene Protective Group)

Xylitol (1.0 mol-152 g) is slurried with 500 ml 2-propanol, and mixedwith dimethoxypropane (3.0 mol-312 g). Following addition of 6 g H₂SO₄in 100 ml 2-propanol, the mixture is heated to 50° C. After 30 minuteseverything has dissolved. Sufficient concentrated ammonia is added toadjust the reaction mixture to a pH of about 8. The solvent is removedin a rotary evaporator, and the residue taken up in hexane and cooled to−20° C. White crystals precipitate, which are sucked up and used for thephosphorylation.

Empirical formula: C₁₁H₁₉O₅ (MW 231.27)

calculated: C, 57.13; H, 8.28; O, 34.59.

measured: C, 57.01; H, 8.27; O, -.

Structural Formulae of Some Sugar Alcohols:

C₄H₁₀O₄ meso-Erythritol D-Threitol L-Threitol

C₅H₁₂O₅ Adonite (Ribitol) D(+)-Arabitol L(−)-Arabitol Xylitol

C₆H₁₄O₆ Dulcite (Galactit. D-Mannitol D-Sorbitol

1,2; 3,4-diisopropylidene-5-benzyl-D-mannitol (General Example for theUse of Trityl Protective Groups Combined with Benzyl Protective Groups)

Starting with 1,2; 3,4; 5,6-triisopropylidene-D-mannitol (MW 302.36),which is prepared analogously to the xylitol derivative, one obtains—bymeans of carefully splitting off the protective group—a1,2,3,4-diisopropylidene-D-mannitol yield of about 30%.Triisopropylidene-D-mannitol (1.0 mol-302 g) is dissolved in 600 mlCH₃OH, to which 15 (5 g) Amberlyst® and 70 g H₂O are then added. Thereaction mixture is heated to 50° C., the solution stirred at thistemperature for 40 minutes (educt, R_(f) 0.9; 1,2; 3,4-derivative, R_(f)0.7; 3,4-derivative, R_(f) 0.1 in CHCl₃/CH₃OH 1:1), cooled to 20° C. andfiltered to 7.5 ml 25% ammonia in 25 ml 2-propanol (pH ˜8). Cooling to4° C. causes the starting product to precipitate, which can thus berecovered (ca. 120 g, ˜40%). The filtrate is rotated in an evaporatorand purified chromatographically on silica gel 60 (Merck, Darmstadt).One obtains 84 g (˜32%) of 1,2:3,4-diisopropylidene-D-mannitol, which isretrieved in crystalline form from hexane.

Empirical formula: C₁₂H₂₂O₆ (MW 262.30)

calculated: C, 54.95; H, 8.45; O, 36.60.

measured: C, 54.89; H, 8.34; O, -.

Reaction of 1,2; 3,4-diisopropylidene-D-mannitol with trityl chlorideand benzyl chloride (General Example for Tritylation and SubsequentAlkylation)

1,2:3,4-diisopropylidene-D-mannitol (0.2 mol-52 g) is dissolved in 300ml toluene, mixed with triethylamine (0.30 mol-30 g) and reflux-boiled.Trityl chloride (MW 278.78; mol-64 g) in 200 ml toluene is addeddropwise, and the mixture reflux-boiled for another 60 minutes (educt,R_(f) 0.7; product, R_(f) 0.90 in CHCl₃/CH₃OH 10:1). The reaction isthen complete. The mixture is cooled to 20° C., precipitatedtriethylamine hydrochloride filtered off, and the filtrate rotated in anevaporator. The residue is taken up in 400 ml THF, mixed with benzylchloride (0.3 mol-38 g) and reflux-boiled. K-tert. butylate (0.25 mol-28g)—dissolved in 200 ml THF—is added dropwise and the reaction mixtureleft to stand for 1 hour (educt, R_(f) 0.90; product, R_(f) 1.00 inCHCl₃/CH₃OH 10:1). Following addition of 300 ml diisopropyl ether, thereaction mixture is extracted with 600 ml H₂O, the upper phase taken offand the solvent removed under vacuum. The residue can be used directly.

Cleavage of the Trityl Protective Group while Retaining the3,4-Isopropylidene Protective Group (General Example)

The oily residue from the preceding reaction (−0.2 mol) is dissolved in600 ml acetone/CH₃OH 1:1, to which 3 ml H₂SO₄ are then added. Themixture is stirred at 40° C. for 40 minutes, which results in completeremoval of the trityl- and the 1,2-isopropylidene protective groups(educt, R_(f) 0.95; product, R_(f) 0.15 in ether). The reaction mixtureis adjusted to pH ˜8, filtered and rotated in an evaporator. The residueis purified chromatographically on silica gel, and crystallized fromhexane.

2-0-benyzl-3,4-isopropylidene-D-mannitol

Empirical formula: C₁₅H₂₄O₆ (MW 312.36)

calculated: C, 61.52; H, 7.74; O, 30.73.

Measured: C, 61.44; H, 7.72; O, -.

As described for example 4c, the isopropylidene protective group can bereintroduced in the 5.6 position. A key intermediate for the synthesisof phosphatidyl-D-mannitol compounds is obtained, namely 2-0-benzyl-3,4;5,6-diisopropylidene-D-mannitol.

Empirical formula: C₁₉H₂₈O₆ (MW 352.42)

calculated: C, 64.75; H, 8.01; O, 27.24.

measured: C, 64.68; H, 7.94; O, -.

Sugar Alcohol Constituents which are Obtained by Splitting Periodate Offa Vicinal Diol and Reducing the Resulting Aldehyde with SodiumBorohydride (General Example)

1,2:3,4-diisopropylidene-D-mannitol (0.2 mol-26 g) is dissolvedaccording to the method of H. Eibl (Chem. Phys. Lipids 28 (1981) 1-5) in200 ml CH₃OH and added to a solution of 0.2 mol sodium metaperiodate in500 ml water. The temperature should not exceed 30° C. The reaction iscomplete after 15 minutes. The pH of the reaction mixture is raised topH=8 with 5 M KOH in water. The precipitated salts are filtered off, andthe aldehyde reduced with sodium borohydride (0.25 mol). One obtainsa >90% yield of 1,2:3,4-diisopropylidene-D-lyxitol, which is extractedwith 600 ml of chloroform. The chloroform phase is rotated in anevaporator and the product crystallized from hexane.

Empirical formula: C₁₁H₁₉O₅ (MW 231.27)

calculated: C, 57.13; H, 8.28; O, 34.59.

measured: C, 57.07; H, 8.21; O, -.

By employing the various alternatives—monoisopropylidene cleavage,periodate cleavage from vicinal diols to produce aldehydes which arethen reduced with sodium borohydride, and the variationtrityl/alkyl—sugar alcohols are obtained that are protected in verydifferent ways. These can be converted by way of acylation orphosphorylation into interesting alkyl, acyl or phosphatidyl compounds.

Preparation of Simple Ester and Ether Derivatives from theOligoglycerols and Sugar Alcohols Portrayed (General Description)

Methods of esterification and etherification, followed by cleavage ofthe protective groups, have been described in various publications. Thearticles listed below include different methods of phosphorylation.These methods can be employed here analogously.

-   Eibl, H.-   Synthesis of glycerophospholipids-   Chem. Phys. Lipids 26 (1980) 405-429-   Eibl, H.-   Phospholipid Synthesis-   In: Liposomes: From Physical Structure to Therapeutic Applications    (C. G. Knight, editor) Elsevier, Amsterdam (1981) 19-50-   Eibl, H. and Kovatchev, S.-   Preparation of phospholipids and of their analogues by    phospholipase D. In: Methods of Enzymology. Vol. 72. Ed. J. M.    Lowenstein, Academic Press, New York (1981) 632-639-   Eibl, H.: Phospholipids als funktionelle Bausteine biologischer    Membranen Angew. Chemie 96 (1984) 247-262-   Eibl, H: Phospholipids as functional constituents of biomembranes    Angew. Chem. Int. Ed. Engl. 23 (1984) 257-271-   Eibl, H. Phospholipid synthesis: Oxazaphospholanes and    dioxaphospholanes as intermediates. Proc. Natl. Acad. Sci. USA    75 (1978) 4074-4077-   Eibl, H. and Wooley, P: Synthesis of enantiomerically pure glyceryl    esters and ethers. I. Methods employing the precursor    1,2-isopropylidene-sn-glycerol. Chem. Phys. Lipids 41 (1986) 53-63-   Eibl. H. and Wooley, P.: Synthesis of enantiomerically pure glyceryl    esters and ethers. II. Methods employing the precursor    3,4-isopropylidene-D-mannitol. Chem. Phys. Kipids 47 (1988) 47-53-   Eibl. H. and Wooley, P.: A general synthetic method for    enantiomerically pure ester and ether lysophospholipids. Chem. Phys.    Lipids 47 (1988) 63-68-   Wooley, P. and Eibl, H.: Synthesis of enantiomerically pure    phospholipids including phosphatidylserine and phosphatidylglycerol.    Chem. Phys. Lipids 47 (1988) 55-62

EXAMPLE 4g Intermediates for the Synthesis of Phospholipids whichContain Sugar Alcohols in the Polar Area

As already described, the introduction via oligoglycerols of substancesin the polar area of phospholipids has a pronounced effect on the bloodcirculation if these substances are used as liposome components. Thesame result can be obtained if, instead of the oligoglycerols, use ismade of sugar alcohols, eg, phosphoric acid esters of D-mannitol,D-lyxitol and D-threitol. These compounds can be introduced withsuitable protective groups (see model H) into phospholipids in themanner described for oligoglycerols. With the derivatives described,coupling with phospholipids again leads to an sn-1 linkage between thephosphoric acid and the sugar alcohol.

D-Mannitol Derivative

3.4-0,0-dibenzyl-D-mannitol is prepared from1,2,6,5-diisopropylidene-D-mannitol by benzylating in the 3.4 positionand splitting off the isopropylidene protective groups. Afterintroducing the isopropylidene protective group in the 1.2 position,tritylation and benzylation of the exposed —OH group, one obtains,following cleavage of the trityl group, the compound XV, which can beincorporated in the polar area of phospholipids.

D-Lyxitol Derivative

1,2-isopropylidene-3,4-0,0-dibenyzl-D-mannitol (see above) is cleavedwith periodic acid and reduced with NaBH₄ to the alcohol XVI. Thiscompound can be incorporated into the polar area of phospholipids.

D-Mannitol Derivative (6 Hydroxyl Groups)

D-Lyxitol Derivative (5 Hydroxyl Groups; from D-Mannitol)

D-Threitol Derivative (4 Hydroxyl Groups; from D-Mannitol)

Model H: Polyhydric Alcohols with at Least 4 Hydroxyl Groups forIncorporation into the Polar Area of Phospholipids

D-Threitol Derivative

The compound XVI is converted by way of benzylation into1,2-isopropylidene-3,4,5-0,0,0-tribenyzl-D-lyxitol. After splitting offthe isopropylidene protective group, periodic acid cleavage andreduction to the alcohol, one obtains XVII. This compound can beincorporated into the polar area of phospholipids in the usual way.

Phospholipids which Contain Oligoglycerols in the Polar Area

In earlier publications we have described how phospholipids can beeasily prepared from diacyl glycerols with saturated and unsaturatedfatty acid chains, with two identical or two different fatty acid chains(DE 32 39 817 Ar; P. Woolley et al. Chem. Phys. Lipids 47 (1988) 55-62;H. Eibl et al., Chem. Phys. Lipids 47 (1988) 63-68). It is also possibleto use acyl/alkyl or alkyl/acyl glycerols as starting product. However,phospholipids which contain dialkyl glycerols are metabolicallyextremely stable and resorption is negligible.

Basically, the compounds referred to can be prepared according to twodifferent methods. This derives from the fact that a phosphoric aciddiester is to be prepared from two alcohols, R₁—OH and R₂—OH.

The R₁—OH alcohols are alcohols which contain a glycerol backbone withtwo fatty acid chains and a free hydroxyl group. They can also have justone fatty acid chain and an additional protective group, usually benzyl,for the preparation of monoacrylic phospholipids; R₁—OH can, however,also stand for an alcohol with a simple alkyl group with one or two cisdouble bonds.

The R₂—OH alcohols are alcohols which have so far been designated as G₂,G₃ and G₄ in the text. They are described by the structural formulae IIIand V (for G₂), VII and VIII (for G₃) and X to XIV (for G₄). In likemanner, use can also be made of the sugar alcohol derivatives XV toXVII.

Model G describes how two alcohols R₁—OH and R₂—OH can be utilized toprepare good yields of phosphoric acid diesters.

Where, for example, R₁=1,2-dipalmitoyl-sn-G and R₂=formula XI, thefollowing structure is obtained after removal of the protective groups:

Model G: Phosphoric Acid Diester with the Formula R₁O—PO⁻ ₃—R₂; Na⁺

Phosphorus oxychloride is used as phosphorylation agent. From the twoalcohols R₁OH and R₂OH to be linked via phosphate, one first of allprepares the corresponding phosphoric acid dichloride; this is reactedin each case with the other alcohol to phosphoric acid monochloride.Slightly acid hydrolysis then leads to the phosphoric acid diesters,which, after the protective groups have been split off, form, eg, thesalt XVIII, 1,2-dipalmitoyl-sn-glycero-3-phospho-G₁-G₂-G₃-G₄; Na⁺.

The following list of examples can be extended at will by usingdifferent combinations of fatty acid chains or by introducing additionalfatty acids, both of synthetic and natural origin. If necessary in orderto obtain particular properties, the phosphatidyl-oligoglycerols cancontain additional alkyl chains or fatty acid residues in theoligoglycerol part.

The oligoglycerol-based methods described here can thus be used andmodified in manifold ways in order to vary and influence the propertiesof liposomes. By analogy with the hexadecylphosphocholines and theerucylphosphocholines, however, these substances may also be importantbiologically active molecules which influence signal transduction andthus functional pathways in the cells.

Examples of phospho-G₁-G₂Compounds1. 1,2-dipalmitoyl-sn-glycero-3-phospho-G₁-G₂;

Na⁺ salt: C₄₁H₈₀NaO₁₂P (819.04)

2. 1,2-dimyristoyl-sn-glycero-3-phospho-G₁-G₂;

Na⁺ salt: C₃₇H₂₂NaO₁₂P (762.93)

3. 1,2-distearoyl-sn-glycero-3-phospho-G₁-G₂;

Na⁺ salt: C₄₅H₈₈NaO₁₂P (875.14)

4. 1-palmitoyl-2-lauroyl-sn-glycero-3-phospho-G₁-G₂;

Na⁺ salt: C₃₇H₇₂NaO₁₂P (762.93)

5. 1-stearoyl-2-lauroyl-sn-glycero-3-phospho-G₁-G₂;

Na⁺ salt: C₃₉H₇₆NaO₁₂P (790.98)

6. 1,2-dioleoyl-sn-glycero-3-phospho-G₁-G₂;

Na⁺ salt: C₄₅H₈₄NaO₁₂P (871.11)

7. 1,2-dierucyl-sn-glycero-3-phospho-G₁-G₂;

Na⁺ salt: C₅₃H₁₀₀NaO₁₂P (983.32)

8. 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-G₁-G₂;

Na⁺ salt: C₄₅H₈₆NaO₁₂P (873.13)

9. 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-G₁-G₂;

Na⁺ salt: C₄₃H₈₂NaO₁₂P (845.07)

10. 1-stearoyl-2-myristoyl-sn-glycero-3-phospho-G₁-G₂;

Na⁺ salt: C₄₁H₈₀NaO₁₂P (819.04)

11. 1-stearoyl-2-palmitoyl-sn-glycero-3-phospho-G₁-G₂;

Na⁺ salt: C₄₃H₈₄NaO₁₂P (847.09)

12. 1-myristoyl-sn-glycero-3-phospho-G₁-G₂;

Na⁺ salt: C₂₃H₄₆NaO₁₁P (552.57)

13. 1-palmitoyl-sn-glycero-3-phospho-G₁-G₂;

Na⁺ salt: C₂₅H₅₀NaO₁₁P (580.62)

14. 1-stearoyl-sn-glycero-3-phospho-G₁-G₂;

Na⁺ salt: C₂₇H₅₄NaO₁₁P (608.68)

15. Erucyl-phospho-G₁-G₂;

Na⁺ salt: C₂₈H₅₆NaO₈P (574.71)

16. Octadecyl-phospho-G₁-G₂;

Na⁺ salt: C₂₄H₅₀NaO₈P (520.62)

17. Hexadecyl-phospho-G₁-G₂;

Na⁺ salt: C₂₂H₄₆NaO₈P (492.56)

18. Tetradecyl-phospho-G₁-G₂;

Na⁺ salt: C₂₀H₄₂NaO₈P (464.51)

19. Oleyl-phospho-G₁-G₂;

Na⁺ salt: C₂₄H₄₈NaO₈P (518.60)

20. 1-O-octadecyl-2-O-methyl-sn-glycero-3-phospho-G₁-G₂;

Na⁺ salt: C₂₈H₅₈NaO₁₀P (608.72)

Examples of phospho-G₁-G₇-G₃ Compounds1. 1,2-dipalmitoyl-sn-glycero-3-phospho-G₁-G₂-G₃;

Na⁺ salt: C₄₄H₈₆NaO₁₄P (893.12)

2. 1,2-distearoyl-sn-glycero-3-phospho-G₁-G₂-G₃;

Na⁺ salt: C₄₈H₉₄NaO₁₄P (949.22)

3. 1-palmitoyl-2-lauroyl-sn-glycero-3-phospho-G₁-G₂-G₃;

Na⁺ salt: C₄₀H₇₈NaO₁₄P (837.01)

4. 1-stearoyl-2-lauroyl-sn-glycero-3-phospho-G₁-G₂-G₃;

Na⁺ salt: C₄₂H₈₂NaO₁₄P (865.06)

5. 1,2-dioleoyl-sn-glycero-3-phospho-G₁-G₂-G₃;

Na⁺ salt: C₄₈H₅₀NaO₁₄P (945.19)

6. 1,2-dierucyl-sn-glycero-3-phospho-G₁-G₂-G₃;

Na⁺ salt: C₅₆H₁₀₆NaO₁₄P (1057.40)

7. 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-G₁-G₂-G₃;

Na⁺ salt: C₄₈H₉₂NaO₁₄P (947.21)

8. 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-G₁-G₂-G₃;

Na⁺ salt C₄₆H₈₈NaO₁₄P (919.148)

9. 1-stearoyl-sn-glycero-3-phospho-G₁-G₂-G₃;

Na⁺ salt: C₃₀H₆₀NaO₁₃P (682.76)

10. Erucyl-phospho-G₁-G₂-G₃;

Na⁺ salt: C₃₁H₆₂NaO₁₀P (648.79)

11. Octadecyl-phospho-G₁-G₂-G₃;

Na⁺ salt: C₂₇H₅₆NaO₁₀P (594.69)

12. Hexadecyl-phospho-G₁-G₂-G₃;

Na⁺ salt: C₂₅H₅₂NaO₁₀P (566.64)

13. 3-O-octadecyl-2-O-methyl-sn-glycero-1-phospho-G₁-G₂-G₃;

Na⁺ salt: C₃₁H₆₄NaO₁₂P (682.80)

Examples of phospho-G₁-G7-G₃-G₄ Compounds1. 1,2-dipalmitoyl-sn-glycero-3-phospho-G₁-G₂-G₃-G₄;

Na⁺ salt: C₄₇H₉₂NaO₁₆P (967.20)

2. 1,2-distearoyl-sn-glycero-3-phospho-G₁-G₂-G₃-G₄;

Na⁺ salt: C₅₁H₁₀₀NaO₁₆P (1023.30)

3. 1-stearoyl-2-lauroyl-sn-glycero-3-phospho-G₁-G₂-G₃-G₄;

Na⁺ salt: C₄₅H₈₈NaO₁₆P (939.14)

4. 1,2-dioleoyl-sn-glycero-3-phospho-G₁-G₂-G₃-G₄;

Na⁺ salt: C₅₁H₉₆NaO₁₆P (1019.27)

5. 1,2-dierucyl-sn-glycero-3-phospho-G₁-G₂-G₃-G₄;

Na⁺ salt: C₅₉H₁₁₂NaO₁₆P (1131.48)

6. 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-G₁-G₂-G₃-G₄;

Na⁺ salt: C₅₁H₉₈NaO₁₆P (1021.29)

7. Erucyl-phospho-G₁-G₂-G₃-G₄;

Na⁺ salt: C₃₄H₆₈NaO₁₂P (722.87)

Examples of phospho-sn-G₁ Compoundssn-1-G₁-G₂:1. 1,2-dipalmitoyl-sn-glycero-3-phospho-sn-1-G₁-G₂;

Na⁺ salt: C₄₁H₈₀NaO₁₂P (819.04)

2. 1,2-distearoyl-sn-glycero-3-phospho-sn-1-G₁-G₂;

Na⁺ salt: C₄₅H₈₈NaO₁₂P (875.14)

3. 1-stearoyl-2-lauroyl-sn-glycero-3-phospho-sn-1-G₁-G₂;

Na⁺ salt: C₃₉H₇₆NaO₁₂P (790.98)

4. 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-sn-1-G₁-G₂;

Na⁺ salt: C₄₅H₈₆NaO₁₂P (873.13)

sn-1-G₁-G2-G3:1. Dipalmitoyl-sn-glycero-3-phospho-sn-1-G₁-G₂-G₃;

Na⁺ salt: C₄₄H₈₆NaO₁₄P (893.12)

2. 1,2-distearoyl-sn-glycero-3-phospho-sn-1-G₁-G₂-G₃;

Na⁺ salt: C₄₈H₉₄NaO₁₄P (949.22)

sn-1-G₁-G7-G₃-G₄:1. 1,2-dipalmitoyl-sn-glycero-3-phospho-sn-1-G₁-G₂-G₃-G₄;

Na⁺ salt: C₄₇H₉₂NaO₁₆P (967.20)

2. 1,2-distearoyl-sn-glycero-3-phospho-sn-1-G₁-G₂-G₃-G₄;

Na⁺ salt: C₅₁H₁₀₀NaO₁₆P (1023.30)

Examples of Linkages with Sugar Alcohols

Phospho-D-Mannitol Compounds

1. 1,2-dipalmitoyl-sn-glycero-3-phospho-D-mannitol;

Na⁺ salt: C₄₁H₈₀NaO₁₃P (835.03)

2. 1,2-distearoyl-sn-glycero-3-phospho-D-mannitol;

Na⁺ salt: C₄₅H₈₃NaO₁₃P (891.13)

3. 1-palmitoyl-2-lauroyl-sn-glycero-3-phospho-D-mannitol;

Na⁺ salt: C₃₇H₇₂NaO₁₃P (788.92)

4. 1-stearoyl-2-lauroyl-sn-glycero-3-phospho-D-mannitol;

Na⁺ salt: C₃₉H₇₆NaO₁₃P (806.97)

5. 1-stearoyl-2-myristoyl-sn-glycero-3-phospho-D-mannitol;

Na⁺ salt: C₄₁H₈₀NaO₁₃P (835.03)

6. 1-stearoyl-sn-glycero-3-phospho-D-mannitol;

Na⁺ salt: C₂₇H₅₄NaO₁₂P (624.67)

7. Octadecyl-phospho-D-mannitol;

Na⁺ salt: C₂₄H₅₀NaO₉P (536.61)

8. 1-O-octadecyl-2-O-methyl-sn-glycero-3-phospho-D-mannitol;

Na⁺ salt: C₂₈H₅₈NaO₁₁P (624.71)

Phospho-D-Lyxitol Compounds

1. 1,2-dipalmitoyl-sn-glycero-3-phospho-D-lyxitol;

Na⁺ salt: C₄₀H₇₈NaO₁₂P (805.00)

2. 1,2-distearoyl-sn-glycero-3-phospho-D-lyxitol;

Na⁺ salt: C₄₄H₈₆NaO₁₂P (861.10)

3. 1-palmitoyl-2-lauroyl-sn-glycero-3-phospho-D-lyxitol;

Na⁺ salt: C₃₆H₇₀NaO₁₂P (758.89)

4. 1-stearoyl-2-lauroyl-sn-glycero-3-phospho-D-lyxitol;

Na⁺ salt: C₃₈H₇₄NaO₁₂P (776.94)

5. 1-stearoyl-2-myristoyl-sn-glycero-3-phospho-D-lyxitol;

Phospho-D-Threitol Compounds

1. 1,2-dipalmitoyl-sn-glycero-3-phospho-D-threitol;

Na⁺ salt: C₃₉H₇₆NaO₁₁P (774.97)

2. 1,2-distearoyl-sn-glycero-3-phospho-D-threitol;

Na⁺ salt: C₄₃H₈₄NaO₁₁P (831.07)

3. 1-stearoyl-2-lauroyl-sn-glycero-3-phospho-D-threitol;

Na⁺ salt: C₃₇H₇₂NaO₁₁P (746.91)

4. 1-stearoyl-2-myristoyl-sn-glycero-3-phospho-D-threitol;

Na⁺ salt: C₃₉H₇₆NaO₁₁P (774.97)

EXAMPLE 4h Phosphorylation Steps (General Directions) Based, by Way ofExample, on the Isolation of1,2-dipalmitoyl-sn-glycero-3-phospho-glycero-glycerol, Na⁺ salt

POCl₃ (0.1 mol-15.3 g) in 15 ml THF is introduced into a three-neckedflask. While vigorously stirring the contents of the ice-cooled flask,one adds—dropwise—1,2-dipalmitoyl-sn-glycerol (0.1 mol-57 g) in 100 mlTHF and, separately, triethylamine (0.11 mol-11 g) in such manner thatthere is always a slight excess of triethylamine compared to1,2-dipalmitoyl-sn-glycerol, which takes up the HCl as it forms. Thetemperature of the reaction mixture should not exceed 16° C. Oncompletion of the addition, the reaction mixture is left to stand for afurther 30 minutes at 16° C. and then subjected to a TLC check to makesure that the reaction is complete (1,2-dipalmitoyl-sn-glycerol, R_(f)0.8; 1,2-dipalmitoyl-sn-glycero-3-phosphoric acid dichloride isconverted by way of methanolysis to the corresponding phosphoric aciddimethyl ester, R_(f) 0.4 in ether.)

The second phosphorylation step is carried out with a protectedoligoglycerol. Here, the conversion with2-0-benzyl-rac-G₁-1,3-0,0-1,2-isopropylidene-rac-G₂ is described. To thereaction mixture with 1,2-dipalmitoyl-sn-glycero-3-phosphoric aciddichloride one adds—dropwise—the above alcohol (0.105 mol-31 g) andtriethylamine (0.13-13 g) in 100 ml THF in such manner that thetemperature of the reaction mixture does not exceed 40° C. After 3 hoursat 40° C. the reaction is complete (starting product phosphoric aciddimethyl ester, R_(f) 0.4; product methyl ester, R_(f) 0.7 in ether).One removes the triethylamine hydrochloride precipitate by filtrationand hydrolyses the reaction mixture, mainly1,2-dipalmitoyl-sn-glycero-3-phospho-2-0-benzyl-rac-glycero-1,3-0,0-1,2-isopropylidene-rac-glycerol-monochloridetogether with incompletely reacted1,2-dipalmitoyl-sn-glycero-3-phosphoric acid dichloride, with 26 gNa₂CO₃ dissolved in 260 ml H₂O. After 4 hours, 400 ml diisopropyl etherare added and the upper phase, which contains the product, rotated in anevaporator until crystals begin to form. 500 ml acetone are now added,and the crystals formed removed under suction at 20° C. The filtratecontains the protected phosphatidylglyceroglycerol, Na⁺ salt (R_(f) 0.6in CHCl₃/CH₃OH/glacial acetic acid/H₂O 600:60:20:5). After removal ofthe solvent, one obtains 48 g crude product, which is heated in 140 mlacetic acid and 60 ml H₂O for 30 minutes to 60-70° C. (cleavage of theisopropylidene protective group). One then adds 500 ml CHCl₃, 600 mlCH₃OH and 400 ml H₂O and shakes thoroughly. The lower CHCl₃ phase iswashed again with 600 ml CH₃OH and 500 ml H₂O, with addition ofsufficient Na₂CO₃ to obtain a pH of 6 in the aqueous phase. The lowerchloroform phase is rotated in an evaporator and the residue taken up in400 ml THF. To remove the benzyl protective group, the solution has 6 gPd/C added to it and is debenzylated in a H₂ atmosphere. The reaction iscomplete after about 4 hours. The catalyst is separated off byfiltration, the solvent removed and the residue (˜30 g) taken up in 100ml CHCl₃. 900 ml acetone are added, and the crystals formed removedunder suction. One obtains a white powder,1,2-dipalmitoyl-sn-glycero-3-phospho-glyceroglycerol, Na⁺ salt, yield:26 g (−32%).

Empirical formula: C_(c) H₈₀NaO₁₂P (MW 819.04)

calculated: C, 60.13; H, 9.85; Na, 2.81; O, 23.44; P, 3.78.

measured: C, 60.01; H, 9.79; Na, -; O, -; P, 3.69.

1,2-dipalmitoyl-sn-glycero-3-phospho-glycero-glycero-glycerol, Na⁺ salt

Empirical formula: C₄₄H₈₆NaO₁₄P (MW 893.12)

calculated: C, 59.17; H, 9.71; Na, 2.57; O, 25.08; P, 3.47.

measured: C, 59.11; H, 9.62; Na, -; O, -; P, 3.45.

1,2-dipalmitoyl-sn-glycero-3-phosoho-qlycero-glycero-glycerol, Na⁺ salt

Empirical formula: C₄₇H₉₂NaO₁₆P (MW 967.20)

calculated: C, 58.37; H, 9.59; Na, 2.38; O, 26.47; P, 3.20.

measured: C, 58.29; H, 9.53; Na, -; O, -; P, 3.19.

To prepare phosphatidyl-oligoglycerols with an aliphatic chain, theso-called lysophosphatidyl-oligoglycerols, one can start with compoundswhich have a benzyl ether group in the sn-2 position of the glycerol,eg, 1-palmitoyl-2-0-benzyl-sn-glycerol, stearoyl-2-0-benzyl-sn-glycerol,1-O-hexadecyl-2-0-benzyl-sn-glycerol,1-O-octadecyl-2-0-benzyl-sn-glycerol etc. We have described thepreparation of these compounds in the publications H. Eibl and P.Woolley, Chem. Phys. Lipids 41 (1986) 53-63 and Chem. Phys. Lipids 47(1988) 55-62. They are phosphorylated in the manner described for thepreparation of 1,2-dipalmitoyl-sn-glycero-3-phospho-glyceroglycerol, Na⁺salt, and reacted with the protected oligoglycerols. The protectivegroups are split off analogously. In the last step, by means ofcatalytic hydrogenolysis with Pd/C (5% on activated charcoal) the benzylgroups are split off both the oligoglycerol part and the glycerol, whichcarries an acyl or alkyl group (see the examples).

Preparation of the alkylphospho-oligoglycerols is easy by comparison, asin this case the corresponding alcohols are reacted according to thegiven phosphorylation pattern. To obtain the unsaturated sorts, however,one must use tetrahydropyranyl instead of benzyl as protective group.

A different strategy altogether is employed to synthesize theunsaturated representatives of this substance group.1,2-dibenzyl-sn-glycerol is phosphorylated in the described manner (see,in addition, German patent application DE 32 39 817), then reacted witha tetrahydropyranyl-protected oligoglycerol. Instead of the hydrolysis,a methanolysis is performed and phosphoric acid triesters obtained, eg,for 2-0-tetrahydropyranyl-rac-G₁-1,3-0,0-1,2-isopropylidene-rac-G₂;

This key intermediate is now subjected to hydrolysis with Pd/C (5% onactivated charcoal). One obtains:

It is now possible to introduce arbitrary unsaturated and saturatedfatty acids at the sn-1 and sn-2 positions of the glycerol molecule.This step is followed, as described in our earlier patent application,by splitting off the methyl group with LiBr and then hydrolysing theisopropylidene and tetrahydropyranyl protective groups in 70% aceticacid at 60 to 70° C. The dioleoyl compounds do not crystallize readilyand must therefore be purified chromatographically; the dierucylcompounds, by contrast, are obtained easily in cristalline form.

The phosphoric acid triester strategy is also recommended for preparingmixed-chain phosphatidyl-oligoglycerols. In this case, as with thesynthesis of lysophosphatidyl-oligoglycerols,1-acyl-2-O-benzyl-sn-glycerols or 1-O-alkyl-2-0-benzyl-sn-glycerols areused as starting products and reacted analogously to1,2-dibenzyl-sn-glycerol. Following catalytic debenzylation one obtainsas intermediates for the G₂ compound:

Unsaturated fatty acids can now be introduced at the free sn-2 position,and the molecule freed from its protective groups as described. It isconvenient that molecules with the fatty acid combination1-palmitoyl-2-oleoyl- or 1-stearoyl-2-oleoyl crystallize readily.

EXAMPLE 5 Additional Properties

As the number of glycerol molecules increases, the number of freehydroxyl groups and thus also the polarity increases. For a proportionof 5% in water, PP-G-PG₄ is the only PP-G-PG_(n) to form a clearisotropic solution. The lipids PP-G-PG₁, PP-G-PG₂ and PP-G-PG₃ dissolvewhen heated above 40° C. in water and form superstructures. When thetemperature falls below 40° C., lamellar structures with differentlypronounced hystereses are formed, which are recognizable on account oftheir anisotropy (birefraction in polarized light). The PP-G-PG₁solution becomes cloudy most quickly, and excess lipid precipitates whenthe solution is left to stand at room temperature. The lamellar phasesof PP-G-PG₂ and PP-G-PG₃ remain stable even at low temperatures (down to4° C.). Whereas the transition from the isotropic to the anisotropicphase at room temperature is recognizable after a few minutes withPP-G-PG₂, it takes several hours for PP-G-PG₃. The differences inpolarity are also evident from the different retention factors (R_(f))in the thin-layer chromatogram on silica gel.

1. A compound having the formula:


2. A compound having the formula: